U.S. patent number 5,766,920 [Application Number 08/370,287] was granted by the patent office on 1998-06-16 for ex vivo activation of immune cells.
This patent grant is currently assigned to Cellcor, Inc.. Invention is credited to Bruce P. Babbitt, Zhengyi J. Zhang.
United States Patent |
5,766,920 |
Babbitt , et al. |
June 16, 1998 |
Ex vivo activation of immune cells
Abstract
Disclosed is a process of activating patient-derived mononuclear
cells by exposing the cells in vitro to substances wo generate
immunoreactive cells. The ex vivo activated cells are then
reinfused into the patient to enhance the immune system to treat
various forms of cancer, infectious diseases, autoimmune diseases
or immune deficiency diseases.
Inventors: |
Babbitt; Bruce P. (Easton,
MA), Zhang; Zhengyi J. (Needham, MA) |
Assignee: |
Cellcor, Inc. (Newton,
MA)
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Family
ID: |
27585790 |
Appl.
No.: |
08/370,287 |
Filed: |
January 6, 1995 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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214400 |
Mar 16, 1994 |
5569585 |
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30607 |
Mar 12, 1993 |
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963486 |
Oct 21, 1992 |
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370287 |
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188262 |
Jan 27, 1994 |
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936730 |
Aug 31, 1992 |
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518322 |
May 7, 1990 |
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68413 |
Jul 1, 1987 |
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696546 |
Jan 30, 1985 |
4716111 |
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407236 |
Aug 11, 1982 |
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370287 |
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300982 |
Sep 6, 1994 |
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975682 |
Nov 13, 1992 |
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747484 |
Aug 19, 1991 |
5192537 |
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681668 |
Apr 8, 1991 |
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405044 |
Sep 11, 1989 |
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903489 |
Sep 4, 1986 |
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595081 |
Mar 30, 1984 |
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Current U.S.
Class: |
435/375 |
Current CPC
Class: |
C07K
16/00 (20130101); C12N 5/0636 (20130101); A61K
2035/124 (20130101); C12N 2501/01 (20130101); C12N
2501/515 (20130101); C12N 2501/82 (20130101); C12N
2502/11 (20130101) |
Current International
Class: |
C07K
16/00 (20060101); C12N 5/06 (20060101); A61K
35/12 (20060101); C12N 005/00 (); C12N
005/08 () |
Field of
Search: |
;435/240.1,240.21,240.2
;935/108 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO 87/05400 |
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Sep 1987 |
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WO |
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WO 88/02774 |
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Apr 1988 |
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WO |
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|
Primary Examiner: Eisenschenk; Frank C.
Attorney, Agent or Firm: Fish & Richardson P.C.
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of U.S. patent
application Ser. No. 08/214,400, filed Mar. 16, 1994, now U.S. Pat.
No. 5,569,585 which is a continuation-in-part of U.S. patent
application Ser. No. 08/030,607, filed Mar. 12, 1993, now
abandoned, which is a continuation-in-part of U.S. patent
application Ser. No. 07/963,486, filed Oct. 21, 1992, now
abandoned.
This application is also a continuation-in-part of U.S. patent
application Ser. No. 08/188,262, filed on Jan. 27, 1994, which is a
continuation-in-part of U.S. patent application Ser. No.
07/936,730, filed on Aug. 31, 1992, now abandoned, which is a
continuation of U.S. patent application Ser. No. 07/518,322, filed
on May 7, 1990, now abandoned, which is a continuation of U.S.
patent application Ser. No. 07/068,413, filed on Jul. 1, 1987, now
abandoned, which is a continuation-in-part of U.S. patent
application Ser. No. 06/696,546, filed on Jan. 30, 1985, now U.S.
Pat. No. 4,716,111, which is a continuation of U.S. patent
application Ser. No. 06/407,236, filed on Aug. 11, 1982, now
abandoned.
This application is also a continuation-in-part of U.S. patent
application Ser. no. 08/300,982 filed on Sep. 6, 1994 which is a
continuation of U.S. patent application Ser. No. 07/975,682, filed
Nov. 13, 1992, now abandoned, which is a divisional of U.S. patent
application Ser. No. 07/747,484, filed Aug. 19, 1991, now U.S. Pat.
No. 5,192,537, which is a continuation of U.S. patent application
Ser. No. 07/681,668, filed Apr. 8, 1991, now abandoned, which is a
continuation of U.S. patent application Ser. No. 07/405,044, filed
Sep. 11, 1989, now abandoned, which is a continuation of U.S.
patent application Ser. No. 06/903,489, filed Sep. 4, 1986, now
abandoned, which is a continuation-in-part of U.S. patent
application Ser. no. 06/595,081, filed Mar. 30, 1984, now
abandoned. The above-referenced applications are incorporated
herein by reference.
Claims
What is claimed is:
1. A process of producing a population of immunoreactive cells
comprising the steps of:
(a) contacting a first sample of mononuclear cells derived from a
patient with OKT3 at or below 37.degree. C. to produce an
OKT3-derived culture supernatant (T3CS);
(b) removing said T3CS from said first sample;
(c) determining the concentration of OKT3 in said T3CS, and if
required, supplementing said T3CS with said OKT3 to achieve a
concentration in the range of 1-25 ng/ml;
(d) providing a second sample of mononuclear cells derived from
said patient; and
(e) contacting said second sample with said T3CS and said OKT3 for
a period of time sufficient to activate said second sample in vitro
to yield a population of immunoreactive cells.
2. The process of claim 1, wherein said OKT3 concentration is in
the range of 10-15 ng/ml.
3. The process of claim 1, wherein step (a) is carried out for 6
hours-7 days.
4. The process of claim 3, wherein step (a) is carried out for 3
days.
5. The process of claim 1, wherein said period of time in step (e)
is 1-30 days.
6. The process of claim 5, wherein said period of time in step (e)
is 5 days.
7. The process of claim 1, further comprising after step (d),
contacting said second sample with a suppressor cell inhibitory
compound.
8. The process of claim 7, wherein said suppressor cell inhibitory
compound is cimetidine, indomethacin, or both cimetidine and
indomethacin.
9. A process of producing a population of antigen-specific
polyclonal T cells comprising the steps of:
(a) contacting a first sample of mononuclear cells derived from a
patient with OKT3 at or below 37.degree. C. to produce a T3CS;
(b) removing said T3CS from said first sample;
(c) determining the concentration of OKT3 in said T3CS, and if
required, supplementing said T3CS with said OKT3 to achieve a
concentration in the range of 0.1-25 ng/ml;
(d) providing a second sample of mononuclear cells derived from
said patient;
(e) contacting said second sample with said T3CS and an antigen for
a period of time sufficient to activate said second sample in vitro
to yield a population of antigen-specific polyclonal T cells.
10. The process of claim 9, wherein said period of time is 1-30
days.
11. The process of claim 9, wherein said antigen is a tumor
antigen.
12. The process of claim 9, wherein said antigen is a bacterial
antigen, viral antigen, or autoantigen.
13. The process of claim 1, further comprising after step (e),
contacting said second sample with IL-2.
14. The process of claim 13, wherein said second sample is
contacted with said IL-2 at 4.degree. C.
15. The process of claim 13, wherein said IL-2 is present in an
amount sufficient to bind to at least 25% of the IL-2 receptors on
the surface of said immunoreactive cells.
16. The process of claim 15, wherein said IL-2 is present in an
amount sufficient to bind to 100% of the IL-2 receptors on the
surface of said immunoreactive cells.
17. The process of claim 1, wherein step (e) is carried out below
37.degree. C.
Description
BACKGROUND OF THE INVENTION
This invention relates to immunotherapy.
Adoptive immunotherapy as an approach to treating cancer has been
evolving rapidly over the past two decades. Beginning in the
1980's, IL-2 and lymphokine-activated killer (LAK) cell-based
immunotherapies were shown to induce tumor responses in patients
with melanoma and renal cell carcinoma (Rosenberg et al., 1985, N.
Eng. J. Med. 313:1485-1492; Rosenberg et al., 1986, Important
Advances in Oncology, DeVita et al., eds, Philadelphia, Lipincott,
p. 55; Schoof et al., 1988, Cancer Res. 48:5007-5010; West et al.,
1987, N. Engl. J. Med. 316:898-905). However, these therapies
require systemic administration of high dose IL-2 which usually is
accompanied by severe toxicity (Siegel et al., 1991, J. Clin.
Oncol. 9:694-704; Margolin et al., 1989, J. Clin. Oncol.
7:486-498). More recently, tumor infiltrating lymphocytes (TIL)
isolated from surgically removed tumors were utilized for adoptive
immunotherapy following ex vivo activation and expansion (Rosenberg
et al., 1986, Science 223:1318-1321; Kradin et al., 1989, Lancet
1:577-580; Speiss et al., 1987, J. Natl. Cancer Instit.
79:1067-1075; Rosenberg et al., 1988, N. Engl. J. Med.
319:1676-1688). Although treatment of patients with TIL has been
shown to achieve somewhat higher tumor response rates relative to
LAK therapy for melanoma (Rosenberg et al., 1986, Science
223:1318-1321; Kradin et al., 1989, Lancet 1:577-580; Spiess et
al., 1987, J. Natl. Cancer Instit. 79:1067-1075; Rosenberg et al.,
1988, N. Engl. J. Med. 319:1676-1688), this type of therapy has not
shown consistent outcomes in clinical trials of other tumors.
Furthermore, the limited availability of sufficient numbers of
lymphocytes from autologous tumor tissue and other problems
associated with long term, high volume cell culture have also
restricted the application of TIL therapy.
SUMMARY OF THE INVENTION
The invention provides a novel, safe, and cost-effective method of
nonspecifically enhancing a cell-mediated immune response to
specific antigens or foreign substances in the body, including
cancer cells. The process involves removing a patient's mononuclear
cells and exposing the cells in vitro to substances which enhance
the immune function of the cells. The ex vivo activated (EVA) cells
are then reinfused into the patient to enhance the patient's immune
responses and to treat various forms of cancer, infectious
diseases, autoimmune diseases, or immune deficiency diseases.
The invention features a process of producing a population of
immunoreactive cells by (a) contacting a sample of mononuclear
cells derived from a patient, e.g., peripheral blood mononuclear
cells (PBMC), with OKT3 at or below 37.degree. C. to produce an
OKT3-derived culture supernatant (T3CS); (b) removing the T3CS from
the sample of patient-derived mononuclear cells; (c) determining
the concentration of OKT3 in the T3CS, and if required,
supplementing the T3CS with additional OKT3 to achieve a
concentration of at least 0.1 ng/ml; (d) providing a second sample
of mononuclear cells derived from the patient; and, (e) contacting
the second sample of cells with the previously-generated T3CS for a
period of time sufficient to yield a population of immunoreactive
cells.
A sample of patient-derived mononuclear cells may contain T cells,
B cells, monocytes and macrophages as well as other immune cells
such as polymorphonuclear leukocytes, neutrophils, eosinophils,
natural killer cells, and stem cells. Mononuclear cells may be
derived from the peripheral blood of the patient, or other sites,
e.g., a tumor or tumor-draining lymph node.
T3CS is a conditioned medium containing a mixture of autologous
cytokines together with OKT3. The autologous cytokines mixture
preferably promotes the growth and differentiation of Thl-type T
cells, rather than Th2-type T cells. The OKT3 which is preferably
in solution phase catalyzes the polyclonal activation of T cells,
while the cytokines act synergistically as co-stimulants to
optimize the overall degree of activation. The presence of both
OKT3 and cytokines prevents the generation of T cells that are
anergic or apoptotic and overcomes signal transduction defects in
mononuclear cells derived from patients with cancer or chronic
infectious diseases.
By the term "immunoreactive cells" is meant polyclonal T cells that
exist in a primed state of activation. Primed cells are
multifunctional, i.e., they possess an enhanced capacity to
proliferate and produce cytokines upon further stimulation. The
primed state of activation of the immunoreactive cells induced by
culture in the OKT3-autologous cytokine mixture can be identified
by measuring the stable biochemical changes, e.g., expression of
growth, differentiation, and activation markers, which occur both
on the cell surface and intracellularly. Immunoreactive cells of
the invention have enhanced immunologic effector function, e.g.,
helper activity (CD4.sup.+ T cells) or cytotoxicity (CD8.sup.+ T
cells), compared to unprocessed patient-derived mononuclear
cells.
Immunoreactive cells have a low spontaneous level of immune
function following processing, but are highly sensitized to respond
to low doses of second signals upon further culture, or in vivo.
The immunoreactive cells of the invention therefore require further
exposure to an immune stimulant, such as an antigen; target cell,
e.g., a tumor cell or virus-infected cell; an inflammatory
molecule; an adhesion molecule; an immune cell, e.g., an accessory
cell; a cytokine; or any combination thereof, to achieve full
immunologic effector function. The immunoreactive cells of the
invention are multifunctional, polyclonally-activated T cells which
have been generated independent of disease-specific antigens
utilizing a mixture of nonspecific lymphocyte activators, i.e.,
autologous cytokines, and a mouse monoclonal antibody, i.e, OKT3,
as synergistic stimulants.
Suppressor cells in a population of patient-derived mononuclear
cells may be inactivated by contacting the second sample with a
suppressor cell inhibitory compound, e.g., cimetidine,
indomethacin, cyclophosphamide, ranitidine, pepsid, or any
combination thereof. Other histamine type-2 receptor blockers may
also be used alone or in combination with the compounds listed
above. Cimetidine and indomethacin are preferably used together at
concentrations of 5.times.10.sup.-5 M (.+-.2-fold) and
0.8.times.10.sup.-8 M (.+-.2-fold), respectively.
The concentration of OKT3 used in the process of the invention is
an amount that promotes activation of the patient's cells, but
leaves minimal surface-bound OKT3 on the activated cell product.
Minimizing the amount of surface-bound OKT3 on the immunoreactive
cells in turn minimizes human anti-mouse antigen (HAMA) immune
responses and rapid clearance of the immunoreactive cells from the
circulation of a patient undergoing therapy with the immunoreactive
cells of the invention. The concentration of OKT3 is preferably
greater than 0.1 ng/ml but less than 25 ng/ml, more preferably 1-25
ng/ml, and most preferably 10-15 ng/ml. In addition to OKT3, any
compound that binds to the T cell receptor or the T cell
receptor-associated CD3 molecule on the cell surface may be used to
stimulate the first sample of patient-derived mononuclear cells to
produce T3CS.
Culture of the first sample with OKT3 is carried out for a period
of time sufficient to produce a mixture of nonspecific lymphocyte
activators capable of promoting the OKT3-catalyzed activation and
differentiation of the second sample of patient-derived mononuclear
cells into a population of immunoreactive cells. The culture period
may range from 1 to 7 days and is preferably 3 days. The length of
culture of the first sample may be adjusted, e.g., prolonged, to
achieve the desired concentration of a nonspecific lymphocyte
activator, e.g., a cytokine, e.g., tumor necrosis factor-alpha
(TNFA) or interleukin-2 (IL-2), in the T3CS.
Culture of the second sample of patient-derived cells with T3CS is
carried out for a period of time sufficient to produce a population
of immunoreactive cells. As discussed above, the immunoreactive
cells are in a primed state of activation, i.e., the cells are no
longer in a resting state but require an additional stimulus, e.g.,
exposure to an antigen or other immune stimuli, to achieve a fully
activated state characterized by enhanced immune function compared
to unprocessed cells. Full activation of immunoreactive cells may
be measured by expression of new cell surface markers, e.g., CD25,
secretion of lymphokines, e.g., IFN.gamma., GM-CSF, or TNF.alpha.,
cellular proliferation, or cellular differentiation into effector
cells, e.g., cytolytic T cells or helper T cells.
Culture of patient-derived mononuclear cells with T3CS may be
carried out for a period of 1 to 30 days, preferably 5 days. In the
absence of antigen, the length of the culture period may be as
short as 1-3 days; in the presence of antigen, the cells may be
co-cultured for a period of 1 to 30 days. The length of culture may
be adjusted, e.g., prolonged, to achieve the desired level of
activation of the immunoreactive cells.
The invention also features a process of producing a population of
immunoreactive cells by (a) providing a first sample of mononuclear
cells derived from a patient; (b) determining the concentration of
Fc-receptor positive accessory cells in the first sample, and if
required, supplementing the first sample with a second sample of
mononuclear cells derived from the same patient to achieve a
concentration of 0.1-50% Fc-receptor positive accessory cells in
the first sample; (c) contacting the first sample with OKT3 at or
below 37.degree. C. to produce a T3cs; (d) removing the T3CS from
the first sample; (e) determining the concentration of OKT3 in the
T3CS, and if required, supplementing the T3CS with OKT3 to achieve
a concentration of at least 0.1 ng/ml; (f) providing a third sample
of mononuclear cells derived from the same patient; (g) contacting
the third sample with T3CS for a period of time sufficient to
activate the third sample in vitro to yield a population of
immunoreactive cells. The Fc-receptor positive cells are preferably
monocytes, but may be granulocytes or dendritic cells. The
concentration of patient-derived monocytes is preferably 0.1-50%,
more preferably 1-30%, more preferably 5-15%, and most preferably
10% of the cells in the sample. The second sample of
patient-derived mononuclear cells may be enriched for monocytes
using cell fractionation techniques known in the art, e.g, panning
or FACS, prior to augmenting the concentration of monocytes in the
first sample.
In another aspect, the process is carried out by: (a) contacting a
first sample of mononuclear cells derived from a patient with OKT3
at or below 37.degree. C. to produce a T3CS; (b) removing the T3CS
from the first sample; (c) determining the concentration of tumor
necrosis factor-alpha (TNF.alpha.) in the T3CS, and if required,
supplementing the T3CS with TNF.alpha. to achieve a concentration
of at least 5 pg/ml; (d) providing a second sample of mononuclear
cells derived from the same patient; (e) inactivating suppressor
cells in the second sample; and, (f) contacting the second sample
with T3CS and for a period of time sufficient to activate the
second sample in vitro to yield a population of immunoreactive
cells. The concentration of TNF.alpha. is preferably in the range
of 100 pg/ml to 100 ng/ml, more preferably in the range of 100
pg/ml to 3000 pg/ml, and most preferably in the range of 500 pg/ml
to 1000 pg/ml. The concentration of a cytokine, e.g., TNF.alpha.,
may be augmented by (1) altering the length of the T3CS-generation
step, (2) supplementing T3CS with purified non-recombinant
cytokine, (3) supplementing T3CS with a recombinant cytokine, or
(4) increasing the concentration of a cytokine-producing
patient-derived mononuclear cell. The T3CS generation step (step
(a)) of the process may be carried out for 1-7 days and is
preferably carried out for 3 days.
In yet another aspect, the process requires after step (b),
determining the concentration of both TNF.alpha. and OKT3, and if
required, supplementing the T3CS with TNF.alpha. to achieve a
concentration of at least 5 pg/ml TNF.alpha. and with OKT3 to
achieve a concentration of at least 0.1 ng/m OKT3.
The levels o
The levels of interferon-gamma (IFN-.gamma.), interleukin-4 (IL-4),
and interleukin-10 (IL-10) in T3CS may be adjusted to achieve
optimal generation of a particular type of immunoreactive cell,
e.g., a Thi-type T cell. Accordingly, the process may include the
steps of: (a) contacting a first sample of mononuclear cells
derived from a patient with OKT3 at or below 37.degree. C. to
produce a T3CS; (b) removing the T3CS from the first sample; (c)
determining the concentration of IFN-.gamma., IL-4, and IL-10 in
the T3CS, and if required, adjusting the concentration of
IFN-.gamma. to at least 500 pg/ml, the concentration of IL-4 to
less than or equal to 20 pg/ml, and the concentration of IL-10 to
less than or equal to 20 pg/ml; (d) providing a second sample of
mononuclear cells derived from the same patient; (e) inactivating
suppressor cells in the second sample; and, (f) contacting the
second sample with T3CS and for a period of time sufficient to
activate the second sample in vitro to yield a population of
immunoreactive cells.
The process of the invention may also include a step in which the
state of activation of the immunoreactive cells is evaluated prior
to administration of the cells to the patient. The cells may be
evaluated phenotypically or functionally, i.e., by measuring the
expression of a cell surface marker indicative of cell activation
and/or differentiation or by measuring cell proliferation in
response to an additional immune stimulus, e.g, antigen or phorbol
myristate acetate (PMA). For example, following cell processing
according to the invention, the number of CD25-positive cells in
the population of immunoreactive T cells may be measured and the
cells discarded if the number is less than 10% of the total number
of T cells in the population. Preferably the number of CD25.sup.+
cells is at least 20% of the total number of T cells in the
population.
The level of activation of the immunoreactive cells may also be
evaluated by measuring proliferation in response to further
stimulation by immune stimulants. The cells are discarded if the
level of proliferation, e.g., the amount of .sup.3 H-thymidine
incorporated into cellular DNA, is less than twice the level of
proliferation of a sample of unprocessed mononuclear cells. The
level of proliferation of the immunoreactive cells is at least
twice that of unprocessed cells and preferably is 5-fold greater
than that of unprocessed patient-derived cells. If necessary, the
level of activation of the immunoreactive cells may be adjusted,
e.g., to a higher level of activation, by altering the length of
the patient-derive mononuclear cell-T3CS co-culture period to
generate the immunoreactive cells, or alternatively, by adding
fresh T3CS.
The mixture of nonspecific autologous lymphocyte activators and
OKT3, i.e., T3CS, preferably contains: interleukin-1-alpha
(IL-1.alpha.), interleukin-1-beta (IL-1.beta.), interleukin-6
(IL-6), interleukin-8 (IL-8), TNF.alpha., tumor necrosis
factor-beta (TNF.beta.), interferon-gamma (IFN.gamma.), granulocyte
macrophage-colony stimulating factor (GM-CSF), monocyte/macrophage
colony stimulating factor (M-CSF) and OKT3. In preferred
embodiments, T3CS contains 12.7 ng/ml (.+-.10-40%) OKT3 in addition
to autologous cytokines in the following amounts (.+-.10%-40%):
IL-1.alpha. (105 pg/ml), IL-162 (1433 pg/ml), IL-6 (808 pg/ml),
IL-8 (213 ng/ml), TNF.alpha. (570 pg/ml), TNF.beta. (171 pg/ml),
IFN.gamma. (14350 pg/ml), M-CSF (1193 pg/ml), and GM-CSF (840
pg/ml). IL-2, interleukin-3, IL-4, interleukin-7, IL-10, IL-12, T
cell growth factor-beta (TGF.beta.), and granulocyte-colony
stimulating factor may each be present in T3CS at a concentration
of less than 20 pg/ml. Preferably, these cytokines are present at a
concentration of less than 5 pg/ml. At least a 20% increase in the
number of CD25.sup.+ T cells in the first sample of patient-derived
mononuclear cells following a T3CS-generation culture compared to a
sample of uncultured mononuclear cells is predictive of sufficient
production of autologous cytokines.
To avoid differentiation of the patient-derived mononuclear cells
into lymphokine-activated killer (LAK) cells, the concentration of
IL-2 must be less than 100 units/ml. The concentration of IL-2 in
T3CS is preferably less than 50 units/ml, and more preferably in
the range of 10-20 units/ml, and most preferably in the range of
1-5 ng/ml (1 unit of IL-2 is approximately equal to 250 pg/ml).
To generate optimal levels of antigen-independent nonspecific
lymphocyte activators, production of T3CS is preferably carried out
at a temperature greater than 29.degree. C. but less than
37.degree. C., e.g., 35.degree. C., for a period of 2 days.
Co-culture of patient-derived mononuclear cells with T3CS to
generate the immunoreactive cells may also be carried out at
sub-physiologic temperature, e.g., a temperature greater than
29.degree. C. but less than 37.degree. C.
Following the incubation of cells with T3CS, the cells may be
removed from the T3CS and contacted with IL-2, preferably in an
amount which is sufficient to bind to at least 25% of the IL-2
receptors on the surface of the immunoreactive cells; more
preferably, the amount of IL-2 is sufficient to saturate the IL-2
receptors on the surface of the immunoreactive cells. Contacting
the immunoreactive cells with IL-2 is preferably done at 4.degree.
C., e.g., during storage or delivery of the cells prior to
administration to the patient.
The invention also features a process of producing a population of
antigen-specific polyclonal T cells by (a) contacting a first
sample of mononuclear cells derived from a patient with OKT3 at or
below 37.degree. C. to produce a T3CS; (b) removing the T3CS from
the first sample; (c) determining the concentration of OKT3 in the
T3CS, and if required, supplementing the T3CS with additional OKT3
to achieve a concentration of at least 0.1 ng/ml; (d) providing a
second sample of mononuclear cells derived from the same patient;
(e) contacting the second sample with T3CS and an antigen for a
period of time, e.g., 1-30 days, sufficient to activate the second
sample in vitro to yield a population of antigen-specific
polyclonal T cells. The concentration of OKT3 in the T3CS is
preferably 0.1-1 ng/ml. To achieve the desired concentration, T3CS
may be supplemented with OKT3, or OKT3 may be removed from T3CS
using methods known in the art, such as chromatography,
antibody-mediated depletion, or filtration. The antigen may be in
the form of a natural or synthetic peptide, cell extract, a
purified antigen, or a recombinantly expressed antigen and may be a
tumor antigen, bacterial antigen, viral antigen, or
autoantigen.
In another aspect, the invention provides an immunoreactive
mononuclear cell produced by the inventive process. The cell is
preferably a T cell, more preferably a Th1-type T cell. The T cell
preferably expresses at least 10%, more preferably at least 75%,
and most preferably at least 100% more cell-surface CD25 than an
unprocessed mononuclear cell, e.g., a mononuclear cell in a resting
state. The cell of the invention is preferably in a primed state,
i.e., the cell proliferates at a rate that is at least twice that
of an unprocessed T cell when contacted with an immune stimulant.
The invention also includes a mixture of immunoreactive cells
produced by the inventive process at least 75% of which are T
lymphocytes, e.g., Th1-type T cells. The cells of the invention can
be used to treat any condition characterized by sub-optimal immune
responsiveness.
Another aspect of the invention features a process for producing a
mixture of autologous nonspecific lymphocyte activators by
collecting mononuclear cells from the blood of a patient afflicted
with cancer or an infectious disease, inactivating suppressor T
cells in the sample of mononuclear cells, and contacting the
mononuclear cells with a compound that binds to the T cell receptor
or the T cell receptor-associated CD3 molecule at or below
37.degree. C., e.g., 29.degree.-36.degree. C., e.g., 35.degree. C.
for 2 days. The T cell receptor-binding compound may bind to the
alpha chain or beta chain of a T cell receptor (or alternatively to
the gamma or delta chain). The CD3-binding compound is preferably
soluble OKT3, but may be any ligand that binds to the CD3 molecule
on the surface of the cell. The cells may be contacted with the
CD3-binding compound in the absence or in the presence of an
antigen; the CD3-binding compound may be removed from the cell
culture supernatant following the production of the mixture of
autologous cytokines. In preferred embodiments, the mixture
contains autologous cytokines in the following amounts
(.+-.10%-40%): IL-1.alpha. (105 pg/ml), IL-1.beta. (1433 pg/ml),
IL-6 (808 pg/ml), IL-8 (213 ng/ml), TNF.alpha. (570 pg/ml),
TNF.beta. (171 pg/ml), IFN.gamma. (14350 pg/ml), M-CSF (1193
pg/ml), and GM-CSF (840 pg/ml), either in the presence or absence
of 12.7 ng/ml (.+-.10-40%) OKT3.
The invention also includes the immunoreactive cells of the
invention together with a pharmaceutically acceptable carrier or
diluent for patient administration.
In yet another aspect, the invention features a method of treating
a tumor or a viral pathogen in a patient by administering to the
patient the immunoreactive cells of the invention. A suppressor
cell inhibiting compound, e.g., cimetidine, indomethacin, or both,
may be concurrently administered to the patient. The method may be
used to treat any type of cancer including both solid tumors and
hematologic tumors, e.g., renal cell carcinoma, breast carcinoma,
prostate carcinoma, colo-rectal carcinoma, pancreatic carcinoma,
ovarian carcinoma, melanoma and non-small cell carcinoma of the
lung as well as leukemias and lymphomas. The methods and
compositions of the invention represent a promising approach to
tumors not treatable by conventional forms of therapy such as
chemotherapy, radiation therapy, or surgery.
Mononuclear cells taken from a patient afflicted with a complex
chronic viral disease may also be processed according to the
invention to yield immunoreactive cells which can then be returned
to the patient to augment the patient's immune response to the
pathogen. Patients infected with pathogenic viruses, e.g.,
hepatitis B virus, hepatitis C virus, recurrent herpesvirus (herpes
simplex virus, varicella zoster virus, cytomegalovirus), papilloma
virus, Epstein Barr viurs and HIV (HIV-1 and HIV-2), may be treated
in this manner.
Other features and advantages of the invention will be apparent
from the following detailed description, and from the claims.
BRIEF DESCRIPTION
The figures will first be briefly described.
FIG. 1A is a flow cytometric histogram showing the level of cell
surface CD25 on T lymphocytes cultured in culture media alone.
FIG. 1B is a flow cytometric histogram showing the level of cell
surface CD25 on T lymphocytes activated by OKT3 alone.
FIG. 1C is a flow cytometric histogram showing the level of cell
surface CD25 on T lymphocytes activated with 25% (vol/vol) T3CS
(patient #1).
FIG. 1D is a flow cytometric histogram showing the level of cell
surface CD25 on T lymphocytes activated with 25% (vol/vol) T3CS
(patient #2).
FIG. 1E is a flow cytometric histogram showing the level of cell
surface CD25 on T lymphocytes activated with 25% T3CS (patient
#3).
FIG. 2A is a bar graph showing cell surface CD25 (IL-2 receptor)
expression of peripheral blood mononuclear cells (PBMC) obtained
from 7 metastatic renal cell carcinoma (mRCC) patients following a
5-day culture with either 25% T3CS, 0KT3-depleted T3CS, or OKT3
alone.
FIG. 2B is a bar graph showing PMA-induced proliferation of PBMC
following a 5-day culture with 25% T3CS, OKT3-depleted T3CS, or
OKT3 alone.
FIG. 3A is a line graph showing the enhanced capacity of chimpanzee
EVA cells (EVA #2) to proliferate upon stimulation with PMA
compared to unprocessed chimpanzee-derived mononuclear cells
(PBMCs).
FIG. 3B is a line graph showing the enhanced capacity of chimpanzee
EVA cells (EVA #3) to proliferate upon stimulation with PMA
compared to unprocessed chimpanzee-derived mononuclear cells
(PBMCs).
FIG. 4 is a line graph showing the enhanced capacity of EVA cells
to proliferate and produce cytokines (IFN.gamma.) upon stimulation
with PMA compared to unprocessed mononuclear cells (PBMC).
FIG. 5 is a line graph showing the enhanced capacity of EVA cells
to proliferate upon stimulation with recombinant IL-2 compared to
unprocessed mononuclear cells (PBMC).
FIG. 6 is a line graph showing the enhancement of PBMC
proliferation in response to a recall antigen (influenza) due to
the addition of irradiated EVA cells as helper cells.
FIG. 7 is a bar graph showing the enhanced cytolytic function of
EVA cells compared to unprocessed mononuclear cells (PBMC).
PROCESSING OF PERIPHERAL BLOOD MONONUCLEAR CELLS
Mononuclear cells to be processed according to the invention can be
obtained from patients, e.g., those afflicted with a malignant
tumor or an infectious disease such as hepatitis B. Peripheral
blood or a mononuclear cell-enriched population of cells (obtained
using known methods, e.g., apheresis) is taken from a patient, and
a portion of the sample is mixed with an anticoagulant, e.g.,
heparin, sodium citrate, ethylenediaminetetraacetic acid, sodium
oxalate. The blood-anticoagulant mixture then is diluted in a
physiologically acceptable solution such as sodium chloride or
phosphate buffered solution. Mononuclear cells are recovered by
layering the blood-anticoagulant composition onto a centrifugation
separation medium such as Ficoll-Hypaque (Pharmacia Corporation) or
Lymphocyte Separation Medium (Litton Bionetics Corporation). The
layered mixture then is centrifuged, and the interface containing
the mononuclear cells is collected and washed.
The suppressor cells in the mononuclear cell population may be
functionally inactivated by contacting the mononuclear cells with
an agent that has a specific affinity for or effect upon suppressor
cells. A particularly suitable composition for inactivating
suppressor cells is an H2 receptor antagonist, such as cimetidine;
a suitable composition for inactivating the suppressor activity of
monocytes is indomethacin. Following suppressor cell inactivation,
the mononuclear cells are suspended in a culture medium containing
a mitogenic compound which binds to the T cell receptor or the T
cell receptor-associated CD3 molecule, e.g., a CD3-binding
compound, e.g., OKT3, to produce T3CS. Cimetidine may be used in
the inventive process to inactivate suppressor cells. The addition
of cimetidine to the medium when PBMC are cultured in the T3CS
resulted in increased activation of T cells as measured by enhanced
proliferative responses of immunoreactive cells upon further
stimulation with PMA (data not shown).
In each sample, the concentration of mononuclear cells can be in
the range of about 0.5-5.0.times.10.sup.6 cells/ml, preferably
1-2.times.10.sup.6 cells/ml. Although any standard tissue culture
medium can be utilized in the process of this invention, the cells
are preferably cultured under serum-free conditions at 37.degree.
C. using a standard tissue culture medium, e.g., AIM V medium
available from Gibco-BRL, Grand Island, N.Y.
Mononuclear cells are generally cultured with OKT3 for a period of
3 days to generate T3CS. The T3CS may be used immediately or stored
frozen and then thawed for use.
The concentration of cytokines, e.g., TNF.alpha., or OKT3
concentration in the T3CS may be measured by any conventional means
such as radioimmunoassay or enzyme-linked immunosorbent assay
(ELISA) using antibodies specific for those components.
EXAMPLE 1
Characterization of T3CS
Generation of T3CS
Peripheral blood mononuclear cells (PBMC) were obtained from
patients with mRCC by leukopheresis and fractionated using ficoll
density separation. The cells from the mononuclear fraction were
cultured at 1.times.10.sup.6 /ml in AIM V medium (Gibco-BRL, Grand
Island, N.Y.) with 25 ng/ml OKT3 (Orthoclone OKT3; Ortho
Pharmaceutical Corporation, Raritan, N.J.) for 3 days in
Lifecell.RTM. bags. To inhibit suppressor cell activity, 50 .mu.M
cimetidine (Tagamet.RTM.; Smith Kline Beecham Pharmaceutical,
Cidra, Pa.) and 10 nM indomethacin (Indocine; Merck Sharp &
Dohme, West Point, Pa.) were also added to the culture medium. At
the end of the culture, the culture bags were centrifuged at
1100.times.g for 20 min at room temperature, and the supernatants
were collected, aliquoted, and stored at -70.degree. C.
Composition of T3CS
The composition of the T3CS was determined by ELISA analysis using
Quantikine kits from R&D Systems (Minneapolis, Minn.) for
IL-.alpha.a, IL-2, 3, 4, 6, 7, 8, TNF.alpha., TNF.beta., GM-CSF,
TGF.beta. and G-CSF, IFN.gamma. kits from Endogen (Boston, Mass.)
and Gibco (Grand Island, N.Y.), IL-10 kits from Biosource
International (Camarillo, Calif.), and IL-1.beta. kits from Cistron
Biotechnology (Pine Brook, N.J.). IL-12 was determined by a
bioassay (phytohemagglutinin (PHA) blast proliferation). The amount
of OKT3 present in T3CS was determined by ELISA using the following
reagents purchased from Vector Laboratories, Inc. (Burlingame,
Calif.): Horse anti-mouse IgG to capture the OKT3 mouse mAb,
biotinylated horse anti-mouse IgG to detect the captured mAb and
ABC reagent consisting of avidin-conjugated horseradish peroxidase
to amplify the signal. Ortho-phenylenediamine dihydrochloride
(Sigma Chemical Co., St. Louis, Mo.) was used as a substrate.
Polyclonal Activation of PBMC
To evaluate EVA cells, cultures were carried out under the same
conditions as used in processing patients-derived cells except on a
smaller scale (5 ml). Excess PBMC obtained from mRCC patients were
isolated by ficoll density gradient and were cultured at
2.times.10.sup.6 /ml for 5 days in complete AIM V medium
(containing 50M cimetidine and 10 nM indomethacin) with either 25%
(vol/vol) T3CS, or various concentrations of OKT3. Cells cultured
with medium alone served as control. After incubation, the cells
were washed and resuspended at a concentration of 10.times.10.sup.6
/ml in "infusion medium" (1% human serum albumin and 0.5% dextrose
in Lactated Ringers solution). The cells were then stored overnight
at 4.degree. C. prior to analysis (to simulate overnight storage
for final QA/QC and shipping to clinical sites).
Phenotypic Analysis of PBMC and EVA Cells
Cell phenotypes were determined by flow cytometry.
Immunofluorescent staining was carried out using mAb specific for
cell surface antigens, e.g., Coulterclones T3-RD1/CD3-RD1, T4-RD1,
T8-RD1 and -FITC, IL-2R-FITC, I3-FITC and 2H4-RD1 (Coulter
Corporation, Hialeah, FL), and Dako UCHL1-FITC (Dako corporation,
Carpintera, Calif.). Appropriate isotype-matched labels were used
as negative controls. Cells were resuspended at 2.times.10.sup.6
/ml in AIM V medium and aliquoted to 100 .mu.l per tube. Two to
four .mu.ls of labeled mabs were added to the cells according to
the manufacturers' recommendation. The cells were then incubated
with the Abs at 4.degree. C. for 30 min, washed once with 500 .mu.l
of cold PBS, and resuspended at a concentration of 4.times.10.sup.5
cells/ml in PBS for immediate analysis using a Coulter Epics
Profile II flow cytometer.
PMA Assay
Proliferative responses to PMA were used to measure the degree of
cell activation. The PBMC or EVA cells were resuspended to
1.times.10.sup.6 cells/ml in AIM V medium with or without 1 ng/ml
PMA, and cultured in triplicate in 96-well flat-bottom plates
(Costar, Cambridge, Mass.) at 37.degree. C. with 5% CO.sub.2 for 48
hours. The cells were pulsed with .sup.3 H-thymidine (1 .mu.Ci
/well) for the last 6 hours of culture, and harvested onto
filtermats. The amount of radioactivity incorporated into the cells
was determined by liquid scintillation counting.
Depletion of OKT3 or Cytokines From T3CS
T3CS was divided into 6 ml aliquots, and incubated with the
appropriate neutralizing antibody (Goat anti-human, from R&D
Systems) at 37.degree. C. for one hour. The amount of antibody used
for depletion was determined by the level of the particular
cytokine known to be present and the antibody activity required for
neutralization as specified by the manufacturer. Specifically, 125
.mu.l of antibody was added for the depletion of IL-1.beta. or
TNF.alpha., 20 .mu.l for IL-6, 60 .mu.l for IFN, 100 .mu.l for
GM-CSF, and 250 .mu.l for IL-8. After incubation, 1 ml of
pre-washed magnetic beads (Advanced Magnetics, Cambridge, Mass.)
conjugated with either rabbit-anti-goat IgG (for cytokine
depletion) or goat-anti-Mouse IgG (for OKT3 depletion) was added to
the samples (2 ml of beads were used for multiple-cytokine
depletion) and incubated with the cells at room temperature for 20
min. The cytokine-bound beads were then removed by placing the tube
in a magnetic holder for 5 min. The cytokine-depleted T3CS was
carefully transferred to a fresh labeled tube. The depletions were
monitored by ELISA analysis.
Cytokine Composition of T3CS
As described above, T3CS was generated by culturing PBMC isolated
from mRCC patients with 25 ng/ml OKT3 for 3 days. Aliquots of
randomly selected mRCC patient T3CS (N=33-42) were analyzed for the
presence of 17 different cytokines and OKT3 by ELISA. As shown in
Table 1, T3CS contained a mixture of monokines and lymphokines
including IL-1.alpha., IL-1.beta., IL-6, IL-8, TNF.alpha. and
.beta., IFN.gamma., M-CSF, and GM-CSF. Several other cytokines,
including IL-2, 3, 4, 7, 10, 12, TGF.beta., and G-CSF, were either
undetectable or detected at very low levels in all samples tested.
The cytokine profile indicated that the cells involved in the PBMC
activation process were predominantly monocytes and Th1-type T
cells (Seder et al., 1994, Ann. Rev. Immunol. 12:635-673) or T
cells that had differentiated into Th1-type cytokine producers
during the cultures. Although kinetic studies indicated that
significant amounts of IL-2 (15-433 pg/ml) were present in all
samples of T3CS analyzed during the first 24 hours of the culture,
the level of IL-2 dropped sharply by day 2 (0-132 pg/ml). IL-2
levels became undetectable by day 3 when the culture supernatant
was harvested. The drop in IL-2 is likely to be due to active
consumption of the cytokine during cell culture. The mean OKT3
concentration in T3CS was 12.7 ng/ml. T3CS contains significantly
lower levels of IL-2 than conventional conditioned media, e.g.,
PHA-generated cell supernatants, Concanavalin-A-generated cell
supernatants, or mixed lymphocyte culture supernatants. Cytokine
levels in T3CS from the majority patients fell into acceptable
ranges, i.e., sufficient amounts of the key cytokines were present
to produce immunoreactive cells in subsequent PBMC cultures. T3CS
from a small number of patients contained low levels of certain
cytokines. As discussed above, the concentration of cytokines may
be augmented, if necessary, by carrying out the T3CS generating
step for a longer period of time or alternatively, by adding
recombinant or nonrecombinant cytokines.
TABLE 1 ______________________________________ COMPOSITION OF
CONDITIONED MEDIUM (T3CS) Cytokines: Mean N
______________________________________ IL-1.alpha. (pg/ml) 105 33
IL-1.beta. (pg/ml) 1433 36 IL-6 (pg/ml) 808 39 IL-8 (ng/ml) 213 39
TNF.alpha. (pg/ml) 570 39 TNF.beta. (pg/ml) 171 39 INF.gamma.
(pg/ml) 14350 40 GM-CSF (pg/ml) 840 39 M-CSF (pg/ml) 1193 39 OKT3
(ng/ml) 12.7 42 ______________________________________ IL-2, 3, 4,
7, 10, 12, TGFB and GCSF are below detectable levels (<3-8
pg/ml) in >90% samples tested.
Accessory Cell Requirement for Production of Immunoreactive
Cells
According to the invention, the concentration of patient-derived
monocytes in a sample of patient-derived mononuclear cells, is
preferably 0.1-50%, more preferably 1-30%, more preferably 5-15%,
and most preferably 10%.
To evaluate the role of monocytes in cultures of patient-derived
mononuclear cells, monocytes were depleted from patient-derived
mononuclear cells using the well-known methods of adherence, e.g.,
to plastic plates, or incubation with L-leucine methyl ester.
Incubation of a monocyte-depleted sample of patient-derived
mononuclear cells with T3CS failed to yield the immunoreactive
cells of the invention. In addition, blocking of the Fc receptor on
the surface of Fc-receptor positive accessory cells, e.g., by
adding an excess of soluble human polyclonal IgG to the
patient-derived mononuclear cell-T3CS co-culture, inhibited the
T3CS-catalyzed generation of immunoreactive cells by 93%. These
data indicate that Fc-receptor positive accessory cells, i.e.,
monocytes, granulocytes or dendritic cells, are required to
generate immunoreactive cells using the methods of the invention
when OKT3 is used in solution phase.
If a sample of patient-derived mononuclear cells has a sub-optimal
concentration of accessory cells, the sample of patient-derived
mononuclear cells may be enriched for monocytes, granulocytes, or
dendritic cells.
Role of Soluble OKT3
The OKT3 used in the process of the invention is preferably in
solution phase rather than solid phase. One advantage of using
soluble OKT3 in the inventive process is that soluble OKT3 mediates
a more physiological interaction between Fc-receptor-bearing
accessory cells, e.g., monocytes, and T cells. By forming a bridge
between these cells, a full complement of costimulatory signals is
initiated, thus minimizing the potential for the generation of
incompletely (anergic) or aberrantly (apoptotic) activated T
cells.
Enhancement of OKT3-Induced T Cell Activation by Autologous
Cytokines
The role of cytokines in the induction of T cell activation was
compared to that of OKT3 alone. PBMC from seven mRCC patients were
cultured for 5 days in AIM-V medium containing either 2.5 ng/ml
OKT3 alone or 25% (vol/vol) T3CS that was first depleted of OKT3
and then reconstituted with 2.5 ng/ml of fresh antibody. This
depletion/reconstitution approach was undertaken to assure that the
amount of biologically active OKT3 in the T3CS would be identical
to the OKT3 control. To avoid the bias from a single aliquot of
T3CS, a pool made from three different mRCC patients was used to
stimulate the PBMC from all seven patients.
The level of T cell activation was determined by the expression of
cell surface IL-2 receptor (CD25) as measured by flow cytometry.
FIGS. 1A-1E are representative immunofluorescence histograms that
demonstrate that CD25 expression on T cells stimulated with the
autologous cytokine-OKT3 mixture was significantly higher than that
on T cells stimulated with OKT3 alone.
Primed Activation State of Immunoreactive Cells
The degree of T cell activation was also assessed functionally by
measurement of the proliferative response of EVA cells upon further
stimulation by PMA, a protein kinase C activator.
FIG. 4 shows the results of an experiment in which immunoreactive
cells were contacted with an immune stimulant, e.g., various
concentrations of PMA. In the absence of further stimulation by
PMA, immunoreactive cells displayed very little spontaneous
proliferation or cytokine secretion when cultured at 37.degree. C.
in AIM V medium alone. However, when contacted with 1-10 ng/ml of
PMA, immunoreactive cells (but not unprocessed PBMC) displayed an
enhanced capacity to proliferate and produce IFN.gamma. (as well as
other Th1-type cytokines such as TNF.alpha., TNF.beta., and GM-CSF
(data not shown)). These results are in contrast to those obtained
with mononuclear cells activated by a conventional stimulant, such
as PHA, which possess high levels of spontaneous proliferation and
cytokine production following an initial 5-day activation culture
(data not shown).
This enhanced proliferation and cytokine secretion upon contact
with an immune stimulant and concomitant absence of or low levels
of spontaneous proliferation and cytokine secretion indicates that
the immunoreactive cells of the invention are in a primed state of
activation.
Similarly, an increase in proliferation was observed when EVA cells
were contacted with another immune stimulant, i.e., IL-2 (FIG. 5).
These data confirm the primed nature of the immunoreactive cells
and suggest that the immunoreactive cells of the invention could be
effectively co-administered with a low dose of IL-2.
Enhanced Effector Function of EVA Cells: Cytolytic T Cells
The ability of the T3CS to support the generation of cytotoxic EVA
cells was evaluated. As shown in FIG. 7, cytotoxicity was
determined using a conventional chromium-51 release assay with the
K562 leukemia cell line and allogeneic human renal carcinoma cell
line 769P as targets. As shown in FIG. 7, EVA cells, i.e., T cells
which have been polyclonally-activated independent of
tumor-associated antigens according to the invention, possessed a
greatly enhanced cytotoxicity toward both tumor lines compared to
unprocessed PBMC. These in vitro results suggest that EVA cells are
capable of directly killing tumor cells in vivo.
Enhanced Effector Function of EVA Cells: Helper T Cells
In addition to enhanced cytolytic function, the immunoreactive
cells of the invention were found to have enhanced helper function.
The ability of T3CS to support the generation of EVA cells with
helper cell function was determined by measurement of the ability
of irradiated EVA cells to enhance the proliferative response of
unprocessed mononuclear cells upon stimulation by a recall antigen,
e.g., an influenza antigen. As shown in FIG. 6, EVA cells added to
cultures at low levels (5-10%) provided helper signals to
unprocessed patient-derived mononuclear cells (presumably through
the secretion of Th1-type cytokines) resulting in a significant
increase in proliferation. These in vitro results suggest that EVA
cells are capable of amplifying and broadening their effects in
vivo through the production of cytokines.
The ability of EVA cells to proliferate and to produce a variety of
cytokines (IL-2, GM-CSF, IFN.gamma., TNF.alpha.) in vitro in
response to further stimulation by such agents as PMA and IL-2, as
well as to lyse tumor cell targets, is greatly enhanced compared to
the PBMC from which they were derived. The lowered activation
threshold of the EVA cells exhibited in vitro suggests that once
they are reinfused into patients, they are likely to demonstrate
enhanced responsiveness to immunological signals, such as weakly
immunogenic tumor antigens which normally are non-stimulatory to
unprocessed cells.
Phenotypic Characterization of Immunoreactive Cells
Following short term (5-day) culture of patient-derived mononuclear
cells in the autologous cytokine mixture/OKT3-containing
conditioned medium, the resulting EVA cells were analyzed for the
expression of cell surface antigens. EVA cells expressed enhanced
levels of a variety of activation and/or differentiation markers on
their cell surface including cytokine receptors, e.g., CD25, major
histocompatibility complex (MHC) antigens, e.g., MHC class II,
adhesion molecules/homing receptors, e.g., CD44/Leu8, costimulatory
molecules, e.g., CD28, and markers of primed or memory T cells,
e.g., CD45RO, as shown in Table 5.
Generation of Immunoreactive Cells: Synergistic Effects of Autoloa
Cytokines Plus OKT3
OKT3 was depleted from the T3CS in order to determine the relative
effects of the anti-CD3 monoclonal antibody and the autologous
cytokines on the induction of CD25 expression on the surface of T
lymphocytes (FIG. 2A) and the proliferation of EVA cells upon
further stimulation by PMA (FIG. 2B). Following depletion of OKT3
from the T3CS, there was little or no measurable activation of T
cells, i.e., the autologous cytokines were not capable of
stimulating resting PBMC in the absence of OKT3. However, when mRCC
patient PBMC were cultured with complete T3CS, the cytokines
functioned in synergy with OKT3 resulting in large increases in T
cell activation relative to the levels achieved with OKT3 alone.
Taken together, these results indicate that OKT3 catalyzes the
generation of immunoreactive cells, while the autologous cytokines
function as costimulants to optimize the overall T cell activation
process.
TABLE 5 ______________________________________ EVA CELL PRODUCT
IDENTITY % (+) LYMPHOCYTES EVA PBMC CELLS
______________________________________ CD3(+) - T CELLS 75% 84%
CD4(+) - HELPER T CELLS 48% 60% CD8(+) - CYTOTOXIC T CELLS 20% 25%
CD3(+)/CD25.sup.(+) - ACTIVATED T CELLS 3% 44% (IL2 RECEPTOR -
EARLY STAGE MARKER) CD3(+)/CLASS II(+) - ACTIVATED 13% 42% T CELLS
(MHC-LATE STAGE MARKER) CD3(+)/CD45RO(+) - T CELLS (PRIMED 49% 69%
"MEMORY" CELLS) CD45RO(+)/CD25.sup.(+) 3%LYMPHOCYTES 36% CD3(+)/LEU
8(+) - T CELLS (LYMPH 31% 59% NODE HOMING RECEPTOR) CD3(+)/CD28(+)
- T CELLS (B7/BB1 0.9* 2.3* RECEPTOR - COSTIMULATORY SIGNAL
TRANSDUCTION) CD3(+)/CD44(+) - T CELLS (EARLY 6.0* 8.3* ACTIVATION
MARKER - CELL ADHESION MOLECULE)
______________________________________ *Mean Channel Fluorescence
Intensity N = 19 mRCC Patient PBMC & EVA Cell Products
Immunoreactive Cell Generation Utilizing Various Recombinant
Cytokine-OKT3 Mixtures
Reconstitution experiments were carried out to identify the
cytokines in the T3CS have the greatest effects on T cell
activation. The roles of 5 major cytokines (IL-1.beta., IL-6,
TNF.alpha., IFN-.gamma., GM-CSF) were analyzed utilizing various
combinations of recombinant cytokines together with OKT3. Table 2
shows the results of a series of experiments in which PBMC from 7
mRCC cancer patients were cultured in an allogeneic T3CS, OKT3
alone, or various OKT3-multiple recombinant cytokine mixtures. Five
out of the six cytokines analyzed were found to enhance the level
of stimulation of T cells compared to the level of stimulation seen
with OKT3 alone, as measured by cell surface expression of CD25 on
T cells. This result is consistent with the observation that the
cytokines in T3CS act synergistically with the OKT3 catalyst in the
activation process. Upon analyzing the data from individual mRCC
patients, however, no single cytokine could be identified as the
dominant costimulant across all cultures. In addition, for the
majority of patient PBMC cultures, optimal T cell activation was
achieved only with complete conditioned medium (T3CS). Taken
together, these results indicate that a single recombinant
cytokine, or even a mixture of several recombinant cytokines, is
not able to provide the full complement of costimulatory signals
required for optimal polyclonal activation of T cells. In contrast,
use of a multiple autologous cytokine-enriched conditioned medium,
i.e., T3CS, consistently resulted in the generation of optimally
immunoreactive cells.
TABLE 2
__________________________________________________________________________
Comparison of Conditioned Medium with OKT3 Plus Recombinant
Cytokines on T Cell Activation in mRCC patients % CD25 Positive
Cells mRCC Patient Stimulant Average #1 #2 #3 #4 #5 #6 #7
__________________________________________________________________________
Medium alone 7 6.4 5.4 5.3 5.4 4.9 5.5 14.0 OKT3 2.4 ng/ml 38 18.8
24.7 54.7 28.6 31.7 64.1 46.3 OKT3 + IL1* + TNFa* 41 40.4 37.3 58.1
31.3 36.3 71.8 48.2 OKT3 + IL1 + IFNg* 44 32.3 37.1 59.7 16.4 39.1
71.9 51.9 OKT3 + IL1 + GM-CSF* 42 26.9 38.0 55.9 22.8 38.1 69.9
43.2 OKT3 + IL1 + IL6* 48 43.5 43.2 55.3 26.5 37.5 70.8 54.9 OKT3 +
IL1 + IFNg + GM-CSF 45 33.5 44.4 58.4 21.6 34.8 68.7 41.9 OKT3 +
IL1 + TNFa + IL6 43 20.4 35.0 69.3 24.1 33.7 70.1 44.2 OKT3 + IL1 +
TNFa + GM-CSF 43 26.1 40.7 66.3 21 .8 33.3 67.5 42.2 OKT3 + IL1 +
IFNg + IL6 43 26.5 38.1 65.0 21.8 38.2 68.5 47.3 OKT3 + IL1 + TNFa
+ GM-CSF + IL-6 41 27.6 39.7 61.2 18.8 39.7 73.1 40.8 OKT3 + IL1 +
TNFa + IFNg + IL-6 41 40.1 34.6 55.8 16.3 37.5 69.8 42.1 OKT3 + IL1
+ TNFa + IFNg + IL-6 + GM-CSF 39 26.1 35.2 39.6 16.0 37.4 72.8 43.9
25% Conditioned Medium** 54 53.3 56.4 69.4 57.3 33.2 74.8 33.8
__________________________________________________________________________
PBMC from seven individual mRCC patients were cultured for 5 days
with OKT3 alone, or OKT3 plus various combinations of cytokines, or
with 25% o an allogeneic conditioned medium. The cells were stored
at 4.degree. C. overnight and then analyzed for CD25 expression by
flow cytometry. *Concentration of recombinant cytokines: IL1b = 250
pg/ml, IL6 = 100 pg/ml, TNFa = 100 pg/ml, IFNg = 250 pg/ml, GMCSF =
250 pg/ml. **25% conditioned medium (WP35) contains: OKT3 2.4
ng/ml, IL1b 650 pg/ml, IL6 400 pg/ml, TNFa 131 pg/ml, IFNg 1341
pg/ml, GMCSF 57 pg/ml.
TNF.alpha. Is an Important Co-stimulant for T Cell Activation
Table 3 shows the results from a series of experiments in which the
contributions of various cytokines present in the T3CS to the
overall T cell activation process was analyzed. The level of
induction of CD25 on PBMC cultured in T3CS was compared to that
achieved when cytokines were selectively depleted from aliquots of
the same T3CS preparation.
Despite quantitative differences between the various RCC patient
PBMC tested, depletion of TNF.alpha. from the T3CS consistently
resulted in a reduction of CD25 expression on the EVA cells
generated from all seven cancer patients, indicating that TNFA
serves a key costimulatory function in enhancing OKT3-catalyzed
polyclonal T cell activation.
The effects of the depletion of other cytokines from the T3CS were
highly variable suggesting that the requirement for other
costimulatory signals for optimal T cell activation varies from
patient to patient, and perhaps even from culture to culture
dependent upon the quality of the PBMC isolated at various points
in time from different patients at different times along their
disease course and upon the expression of cytokine receptors on
freshly isolated mononuclear cells.
These results confirm that a single recombinant cytokine, or even a
mixture of several recombinant cytokines, is not able to provide
the necessary costimulatory signals for optimal polyclonal
activation of T cells, and the complete spectrum of autologous
cytokines in T3CS is necessary to generate the immunoreactive cells
of the invention.
TABLE 3
__________________________________________________________________________
Effect of Depletion of Various Cytokines from Conditioned Medium on
T Cell Activation IL-2 Receptor Expression (% of Control) Complete
Cytokine Depleted Patient # Conditioned Media TNF.alpha. IL-1 IL-6
IL-8 GM-CSF IFN.gamma.
__________________________________________________________________________
1 100 84 88 110 78 96 99 2 100 88 76 115 77 63 98 3 100 79 91 86 89
87 103 4 100 85 95 109 85 122 88 5 100 71 92 86 99 93 185 6 100 65
105 92 80 85 140 7 100 68 83 85 117 117 115 Average 100 77 90 98 89
95 118
__________________________________________________________________________
PBMC from seven different mRCC patients were stimulated with 25%
allogeneic conditioned media either complete or depleted with one
of the cytokines listed. The cells were harvested 5 days later and
examined for surface IL2 receptor (CD25) expression by flow
cytometry the next day. Th effect of cytokine depletion is
expressed as % of control which is the total IL2R expression (%
CD25 positive cells x mean channel fluorescence intensity) on cells
activated by the complete, undepleted conditioned media.
T3CS was utilized as a nonspecific stimulant in the ex vivo
generation of polyclonally activated T cells for the treatment of
metastatic renal cell carcinoma. T3CS produced according to the
invention contains a wide variety of monokines and lymphokines
together with low levels of OKT3. OKT3 functions synergistically
with the cytokines in the T3CS. Removal of OKT3 from the T3CS
resulted in a substantial decrease of T cell activation
demonstrating the role of anti-CD3 monoclonal antibody as a
catalyst while the cytokines provide the costimulatory T cell
activation signals.
Conditions in which OKT3 and autologous cytokines function
synergistically in T cell activation have been achieved in
serum-free medium. Serum-free culture is particularly desirable for
the generation of EVA cells used in adoptive immunotherapy.
The claimed process employs aliquots of the T3CS generated in
advance to be used as a stimulant in the secondary cultures. This
approach ensurcomphat a full complement of costimulatory signals
are available at the initiation of each activation culture and
therefore minimizes the probability of generating anergic or
apoptotic T cells. Furthermore, use of the pre-manufactured and
quality-assured autologous cytokine-OKT3 mixture as a stimulant
decreases the dependence upon de novo synthesis of cytokines during
the early stage of the subsequent T cell activation cultures. Such
decreased dependency is especially important under the conditions
such as off-site cell processing that require shipping and storage
of the cells, and when dealing the with cells from patients of
various clinical stages.
PBMC obtained from individual cancer patients, and even PBMC taken
from the same patient at different times, respond differently to
various cytokines. OKT3-induced T cell activation for one patient's
cells may strongly be affected by a given cytokine while cells from
another patient are not affected at all by the same cytokine. This
variation is due both to the inherent diversity of human
lymphocytes and accessory cells, and to the fluctuation caused by
the process prior to the activation such as collection of the cells
by apheresis. overnight storage and shipping may affect accessory
cell activity which in turn affects the dependence of the T cells
on exogenous cytokine present in the culture. Hence, the methods of
the invention require evaluation of various key components of the
T3CS, e.g., OKT3 and TNF.alpha. concentration, and the
concentration of accessory cells, e.g., monocytes. The methods may
also require the evaluation of the activation state, i.e., PMA
responsiveness, of the processed cells to predict their clinical
effectiveness.
IL-2-Independent Generation of Immunoreactive Cells
Utilizing T3CS as a stimulant for mononuclear cells, it is possible
to generate T cells expressing high levels of CD25/IL-2 receptors,
independent of the inclusion of high levels of exogenous IL-2 in
the culture medium. Although low levels of IL-2 are present in both
the T3CS and EVA cultures at early timepoints, there is no
detectable (<6 pg/ml) autologous IL-2 present in the T3CS when
the EVA culture is initiated. Consequently, in contrast to LAK and
TIL cells which are both cultured in high levels of IL-2, the
immunoreactive cells generated by the inventive process are
presumably less dependent upon systemic administration of high dose
IL-2 for therapeutic efficacy. In addition, the lack of IL-2 in the
T3CS allows the production of immunoreactive cells, i.e.,
multifunctional, polyclonal T cells containing both CD8.sup.+
/cytotoxic T cells and a high percentage of CD4.sup.+ /helper
cells, in contrast to PBMC grown in high dose IL-2 which are highly
enriched in CD8+/cytotoxic T cells. Thus, the immunoreactive cells
of the invention have broader functional capacities than PBMC
cultured in IL-2.
If production of T3CS containing low levels, e.g., up to 10
units/ml, of autologous IL-2 is desired to further enhance the
generation of immunoreactive cells, IL-2 production may be
increased and IL-2 consumption simultaneously decreased by
modification of the culture conditions, such as the kinetics and
temperature of the T3CS generation process.
Following processing, the immunoreactive cells may be exposed to
IL-2 at 4.degree. C. such that IL-2 binds to at least 25% of the
cell surface IL-2 receptors. Preferably, the cell surface IL-2
receptors are saturated, i.e., 100% of the IL-2 receptors on the
surface of a cell are bound to IL-2, with IL-2 prior to infusion
into a patient.
Immunoreactive cells with cell surface bound IL-2 are likely to
have an enhanced ability to expand in vivo and a decreased
dependence upon helper cell-mediated IL-2 production, an activity
which may be lacking or depressed in immunosuppressed cancer
patients. In addition, administration of IL-2 to a patient in a
cell-bound form avoids the toxicity and other clinical
complications often associated with intravenous or subcutaneous
co-administration of high dose IL-2 to support cell therapy.
OKT3
Intravenous OKT3 administration, utilized for the suppression of
transplant graft rejection in humans, induces rapid (within 24
hours) removal of a high percentage of CD3.sup.+ T cells from the
circulation due to the presence of the foreign antibody molecules
on the lymphocyte cell surface (Vigeral et al., 1986,
Transplantation 41:730; Chatenoud et al., 1986, J. Immunol.
137:830-838). Excessive amounts of OKT3 bound to an EVA cell
surface may accelerate the clearance of the T cells, resulting in
decreased therapeutic efficacy. The use of T3CS as a stimulant
minimizes this clinical problem because T cell activation can be
achieved with a minimal concentration of OKT3 in the presence of
autologous cytokines compared to the concentration required in the
absence of autologous cytokines.
The use of the T3CS to stimulate PBMC allows a high level of T cell
activation to be catalyzed by a minimal amount of OKT3 (1-4 ng/ml).
The inventive methods leave very little detectable surface-bound
antibody on the activated T cell product, and therefore decrease
the probability of the EVA cells being rapidly removed from the
circulation. In addition, the low level of surface-bound OKT3
allows more CD3-TCR complexes on the surface of the activated T
cells to remain unoccupied so that key molecules such as MHC Class
I and II molecules, tumor antigens, and immunogenic peptides may
bind to and stimulate the T-cell receptor following re-infusion of
the cells into patients. Stimulation of PBMC with higher
concentrations of OKT3 tends to drive the cell culture towards CD8
cell dominance, whereas culture in the autologous cytokine-OKT3
mixture maintains a relatively high CD4/CD8 ratio which is
desirable for the therapeutic function of EVA cells. Finally, the
low level of cell-bound OKT3 reduces the danger of a patient
developing HAMA responses after multiple infusions of EVA
cells.
EXAMPLE 2
Effect of Temperature on Cytokine Production
Conditions under which patient-derived mononuclear cells are
cultured with OKT3 to generate T3CS were analyzed. For the
generation of T3CS, cells were cultured for a period of 1 to 5
days. As shown in table 4, it is possible to manipulate the time
course and/or temperature at which T3CS is generated in order to
selectively increase or decrease the levels of certain autologous
cytokines.
The effect of temperatures ranging from 29.degree. C. to 39.degree.
C. on the production of cytokines was also tested. As shown in
Table 4, higher levels of cytokines were detected in cultures grown
at temperatures of 37.degree. C. or below.
EXAMPLE 3
Production and therapeutic administration of EVA cells
Human peripheral blood cells are collected by apheresis, and the
PBMC are isolated using a Ficoll gradient. The PBMC are incubated
at 37.degree. C. at a concentration of 10.sup.6 cells/ml in AIM V
medium containing cimetidine (5.times.10.sup.-5 mol/l),
indomethacin (10.sup.-8 mol/l), and 25 ng/ml OKT3 (Ortho Biotech,
Raritan, N.J.). The activity of suppressor T cells and of
suppressor monocytes is inactivated by culturing the PBMC with
cimetidine and indomethacin. After three days, the culture
supernatant, i.e., T3CS, is harvested, divided into portions, and
frozen at -80.degree. C. until required for use in the cell
cultures.
A second sample of PBMC is collected from a patient, and suspended
at approximately 2.times.10.sup.6 cells/ml in AIM V medium
containing 25% v/v T3CS, cimetidine (5.times.10.sup.-5 mol/l) and
indomethacin (10.sup.-8 mol/l). The cells are harvested following a
five day culture at 37.degree. C. in a moist-air incubator
containing 5% CO.sub.2, then resuspended at 10.sup.7 cells/ml.
Following overnight storage of the activated cells at 40.degree.
C., approximately 10.sup.9 cells are reinfused intravenously at
room temperature to the patient over 30 min.
If the concentration of OKT3 or TNF.alpha. or other cytokines in
the T3CS are below the preferable ranges, the levels of one or more
of the cytokines may be increased as follows. One way to increase
the amount of a cytokine, e.g., TNF.alpha., is to adjust, e.g.,
increase, the time of culture before harvesting the T3CS.
Alternatively, the concentration of monocytes (which produce
TNF.alpha.) may be increased in the T3CS-generating culture, or the
temperature at which the T3CS-generating step is carried out may be
adjusted to optimize TNF.alpha. production. Another way to increase
the amount of a cytokine is to increase the percentage of T3CS
added to the second sample of patient-derived mononuclear cells,
i.e., by adding greater than 25% T3CS.
The therapeutic methods of the invention which utilize EVA cells
may be used to treat mRCC and other types of metastatic cancers, as
well as infectious diseases, autoimmune diseases, and
immunodeficiency diseases. Clinical outcome may be assessed by such
measures as length of patient survival, quality of life
measurements, changes in any indicators of medical function such as
clinical chemistries, size of tumors, changes in load of virus,
bacteria, fungus, or parasite, toxicity of the therapy, or delay in
time of recurrence of the disease, or other assessments. Clinical
outcome may also be evaluated by monitoring changes in immune
status or time to recurrence of a tumor in a cancer patient.
TABLE 4
__________________________________________________________________________
Temperature/Timecourse Study EC46 AVERAGE OF ALL FIVE PATIENTS
Cytokine Days 39.degree. C. 37.degree. C. 34.degree. C. 33.degree.
C. 32.degree. C. 29.degree. C. Days 39.degree. C. 37.degree. C.
34.degree. C. 33.degree. C. 32.degree. 29.degree.
__________________________________________________________________________
C. 1 521.4 557.8 ND 946.3 ND 831.6 1 183.9 304.7 406.1 758.8 363.1
434.9 IL-2 2 68.9 18.6 ND 441.2 ND 689.3 2 17.4 31.7 257.1 403.8
218.1 381.1 3 6.0 1.3 ND 44.7 ND 228.0 3 3.5 2.5 78.9 53.8 43.4
204.3 5 0.8 3.2 ND 1.8 ND 17.6 5 2.6 2.7 2.8 0.9 2.6 74.8 1 1427.1
1819.5 ND 702.9 ND 592.2 1 569.9 993.1 568.3 593.6 525.5 319.5 TNFa
2 1237.2 1774.2 ND 1735.2 ND 668.1 2 469.6 1036.1 813.1 1634.6
1006.9 707.9 3 1064.4 1318.2 ND 2134.8 ND 1861.5 3 276.9 478.3
672.5 2460.3 633.7 1170.8 5 629.4 830.4 ND 2031.9 ND 2706.6 5 149.7
205.1 231.5 1612.8 188.1 1258.6 1 1031.2 740.0 ND 341.6 ND 198.4 1
422.8 420.7 432.4 311.2 270.8 122.2 IL-6 2 1657.6 1486.4 ND 1100.0
ND 597.6 2 653.9 804.5 751.3 1146.4 690.0 421.5 3 1740.8 1537.6 ND
1581.6 ND 1076.0 3 626.2 741.4 822.8 1579.6 946.1 621.4 5 1879.2
1438.4 ND 1738.4 ND 1448.0 5 542.8 522.2 533.5 1378.8 466.7 709.1 1
292.8 306.4 ND 237.8 ND 233.6 1 416.6 455.1 ND 289.5 ND 270.5
GM-CSF 2 416.8 497.6 ND 351.0 ND 258.5 2 481.9 715.8 ND 530.0 ND
365.1 3 549.3 590.6 ND 506.5 ND 487.4 3 345.6 397.1 ND 975.6 ND
601.7 5 667.2 635.7 ND 685.5 ND 741.4 5 348.2 322.2 ND 669.1 ND
871.8 1 ND ND ND ND ND ND 1 370.3 899.9 497.2 ND 511.7 166.3 gIFN 2
ND ND ND ND ND ND 2 1812.0 3839.3 1463.9 ND 1578.1 272.6 3 ND ND ND
ND ND ND 3 1880.3 5232.8 3101.5 ND 3843.8 350.2 5 ND ND ND ND ND ND
5 931.5 4009.2 3086.2 ND 3192.4 830.7 1 925 782 ND 413 ND 297 1
462.8 795.8 633.3 409.5 563.0 304.2 lL-1.beta. 2 1,916 1,862 ND
1,532 ND 839 2 949.2 1658.0 1435.3 1179.5 1165.3 711.2 3 1,675
1,747 ND 994 ND 453 3 683.6 1498.6 1540.3 944.0 1393.7 542.2 5
1,780 1,653 ND 1,527 ND 1,169 5 535.2 1195.2 1430.0 1371.5 944.0
802.2 1 11.9 13.0 ND 13.9 ND 10.0 1 13.6 19.2 21.3 17.0 20.4 17.5
OKT3 2 9.4 8.7 ND 11.8 ND 10.7 2 19.5 15.9 20.9 15.9 19.7 17.2 3
8.7 6.3 ND 8.8 ND 13.5 3 14.2 11.9 17.4 12.8 17.9 16.2 5 6.0 4.8 ND
6.8 ND 10.9 5 11.7 7.6 12.1 10.0 11.6 15.5
__________________________________________________________________________
EXAMPLE 4
Treatment of Hepatitis B Infection
Polyclonal Activation of PBMC from Chimpanzees with Chronic
Hepatitis B Infection
Viral hepatitis refers to an infection of the liver caused by a
small group of hepatotropic viruses that may be differentiated
serologically. Of the three serotypes, A, B and C; B and C cause
far more serious infections than hepatitis A.
The methods of the invention were used to produce immunoreactive
cells from mononuclear cells derived from chimpanzees infected with
hepatitis B, a primate model of chronic viral infection. The
chimpanzee model was chosen over the woodchuck or mouse model as
the most relevant model of the human disease because chimpanzees
can be experimentally infected with the human hepatitis B
virus.
Mononuclear cells from chimpanzees with chronic hepatitis B were
polyclonally activated with OKT3 to produce a T3CS. A second sample
of mononuclear cells was cultured with T3CS to obtain EVA cells.
Chimpanzee-derived mononuclear cells processed according to the
invention yielded immunoreactive cells. As shown in FIGS. 3A and
3B, the immunoreactive cells were in a primed state of activation,
i.e., they proliferated upon stimulation with PMA whereas
unprocessed chimpanzee-derived mononuclear cells did not. In
addition, the spectrum of phenotypic markers on the chimpanzee EVA
cells was found to be similar to that on EVA cells produced from
patient-derived mononuclear cells (data not shown). These data
indicate that the methods of the invention can be used to generate
immunoreactive cells from animals, e.g., patients, with chronic
viral infections. These immunoreactive cells can then be re-infused
into the patient to augment the patient's immune response to the
pathogenic virus.
EXAMPLE 5
In vivo Use of T3CS
T3CS may also be used as a formulation for the in vivo delivery of
cytokines. T3CS which has been depleted of OKT3 may be encapsulated
in an appropriate slow release matrix, e.g., a liposome or
biocompatible polymer, in order to maintain a critical level of
cytokine over time and to minimize the toxicity associated with
bolus-delivery of cytokines. In vivo delivery of T3CS may also be
targeted to a particular site in the body for optimal
effectiveness.
In vivo administration of T3CS offers several advantages over
conventional cytokine therapy, i.e., administration of one
cytokine. T3CS is a unique mixture of both monokines and
lymphokines which initiate and promote activation and/or
recruitment of several different immune cell types. Thus, T3CS can
induce a full spectrum of immune responses in a patient.
Other embodiments are within the following claims.
* * * * *